The principal goal in this work is the identification and detailed characterization of proteins and peptides at a microscale level using mass spectrometry. For identification of unknown proteins, the MS data are used to query genomic databases to ask the general question, "Do any of the protein sequences present in the data base have expected proteolytic cleavage products with theoretical masses that match the empirically determined masses of the peptides generated from the unknown?" Three mass spectrometric approaches are available for this effort. Matrix Assisted Laser Desorption Ionization (MALDI) with Time-of-Flight (TOF) mass analysis, liquid chromatography (LC) followed by electrospray ionization with mass analysis in an instrument capable of using fragmentation reactions to generate peptide sequences, i.e. LC-MS/MS, and most recently MALDI followed by tandem TOF analysis for the determination of peptide sequences from fragment ion spectra. With this combination of instrumentation, we are confident that, given enough material in a gel band to allow as much as 100 fmole to be available for analysis, a positive identification can be made for a protein that is described in a database. There are several areas of development that are being followed in order to improve protein characterization capabilities. First, we have begun addressing the question of providing sequence information on proteins that are not described in data bases, due to data base error or incompleteness, most frequently associated with organisms having unknown or partially characterized genomes. We are taking the approach we have termed "Complete de novo Sequencing of Peptides". This approach is novel in comparison to the other widely used methods, in which the so-called "sequence tag" for a peptide is found. The sequence tag consists of determining between 2 and 5 amino acid residues from a peptide fragmentation mass spectrum along with the parent mass to search a database. Our approach requires the determination of the amino acid sequence of the entire peptide. This approach is limited to the use of a MALDI tandem TOF, and in that regard is a capability unique at NIH. Recent results, making use of the recently upgraded MALDI tandem TOF instrument, show that our procedure yields very reliable sequences for as many as seven peptides in the range of 8 to 17 residues taken from the tryptic proteolysis of sea urchin proteins. We are able to employ subsequent collision induced dissociation (CID) experiment in the tandem TOF. Under those circumstances, a sequence determined in the first experiment can not only be verified, but also allow Leu residues to be distinguished from Ile. The detailed interpretation of individual fragmentation mass spectra of peptides required for this approach is being implemented using novel software designed and written in this Section. In two closely related areas, we are developing methods both for the identification of two types of post-translational modifications, phosphorylation and disulfide links. The development of both of these methodologies involves the use of multiple mass spectrometry experiments and the use of novel software written in this Section. These two types of problems are complex in a different fashion than those involving sequence determination since the proteins being investigated in these cases must have known sequences. In order to determine the modifications effectively, virtually complete peptide coverage of the proteins must be obtained using digestion and mass spectrometry. Phosphorylation site identification involves first the enrichment/isolation of phosphopeptides using metal affinity chromatography followed by a combination of mass spectrometry. Potential phosphopeptides are found by differences in ion intensities between positive and negative ion MALDI TOF spectra. Phosphorylation sites are then identified from amino acids residues undergoing neutral phosphate losses in using LC-MS/MS or tandem TOF approaches. Identifying the sites of disulfide bridges is done in a somewhat similar fashion. MADLI TOF spectra of proteolytic peptides obtained from reduced and unreduced proteins are compared and peptides in the spectrum of the reduced material that are not present in the unreduced are sequenced. Potential pairing of those peptides with masses that are unique to the unreduced material is then investigated.